The present invention relates to experimentally-generated cold-adapted equine influenza viruses, and particularly to cold-adapted equine influenza viruses having additional phenotypes, such as attenuation, dominant interference, or temperature sensitivity. The invention also includes reassortant influenza A viruses which contain at least one genome segment from such an equine influenza virus, such that the reassortant virus includes certain phenotypes of the donor equine influenza virus. The invention further includes genetically-engineered equine influenza viruses, produced through reverse genetics, which comprise certain identifying phenotypes of a cold-adapted equine influenza virus of the present invention. The present invention also relates to the use of these viruses in therapeutic compositions to protect animals from diseases caused by influenza viruses.
Equine influenza virus has been recognized as a major respiratory pathogen in horses since about 1956. Disease symptoms caused by equine influenza virus can be severe, and are often followed by secondary bacterial infections. Two subtypes of equine influenza virus are recognized, namely subtype-1, the prototype being A/Equine/Prague/1/56 (H7N7), and subtype-2, the prototype being A/Equine/Miami/1/63 (H3N8). Presently, the predominant virus subtype is subtype-2, which has further diverged among Eurasian and North American isolates in recent years.
The currently licensed vaccine for equine influenza is an inactivated (killed) virus vaccine. This vaccine provides minimal, if any, protection for horses, and can produce undesirable side effects, for example, inflammatory reactions at the site of injection. See, e.g., Mumford, 1987, Equine Infectious Disease IV, 207-217, and Mumford, et al., 1993, Vaccine 11, 1172-1174. Furthermore, current modalities cannot be used in young foals, because they cannot overcome maternal immunity, and can induce tolerance in a younger animal. Based on the severity of disease, there remains a need for safe, effective therapeutic compositions to protect horses against equine influenza disease.
Production of therapeutic compositions comprising cold-adapted human influenza viruses is described, for example, in Maassab, et al., 1960, Nature 7,612-614, and Maassab, et al., 1969, J. Immunol. 102, 728-732. Furthermore, these researchers noted that cold-adapted human influenza viruses, i.e., viruses that have been adapted to grow at lower than normal temperatures, tend to have a phenotype wherein the virus is temperature sensitive; that is, the virus does not grow well at certain higher, non-permissive temperatures at which the wild-type virus will grow and replicate. Various cold-adapted human influenza A viruses, produced by reassortment with existing cold-adapted human influenza A viruses, have been shown to elicit good immune responses in vaccinated individuals, and certain live attenuated cold-adapted reassortant human influenza A viruses have proven to protect humans against challenge with wild-type virus. See, e.g., Clements, et al., 1986, J. Clin. Microbiol. 23, 73-76. In U.S. Pat. No. 5,149,531, by Youngner, et al., issued Sep. 22, 1992, the inventors of the present invention further demonstrated that certain reassortant cold-adapted human influenza A viruses also possess a dominant interference phenotype, i.e., they inhibit the growth of their corresponding parental wild-type strain, as well as heterologous influenza A viruses.
U.S. Pat. No. 4,683,137, by Coggins et al., issued Jul. 28, 1987, and U.S. Pat. No. 4,693,893, by Campbell, issued Sep. 15, 1987, disclose attenuated therapeutic compositions produced by reassortment of wild-type equine influenza viruses with attenuated, cold-adapted human influenza A viruses. Although these therapeutic compositions appear to be generally safe and effective in horses, they pose a significant danger of introducing into the environment a virus containing both human and equine influenza genes.
The present invention provides experimentally-generated cold-adapted equine influenza viruses, reassortant influenza A viruses that comprise at least one genome segment of an equine influenza virus generated by cold-adaptation such that the equine influenza virus genome segment confers at least one identifying phenotype of a cold-adapted equine influenza virus on the reassortant virus, and genetically-engineered equine influenza viruses, produced through reverse genetics, which comprise at least one identifying phenotype of a cold-adapted equine influenza virus. Identifying phenotypes include cold-adaptation, temperature sensitivity, dominant interference, and attenuation. The invention further provides a therapeutic composition to protect an animal against disease caused by an influenza A virus, where the therapeutic composition includes a cold-adapted equine influenza virus a reassortant influenza A virus, or a genetically-engineered equine influenza virus of the present invention. Also provided is a method to protect an animal from diseases caused by an influenza A virus which includes the administration of such a therapeutic composition. Also provided are methods to produce a cold-adapted equine influenza virus, and methods to produce a reassortant influenza A virus which comprises at least one genome segment of a cold-adapted equine influenza virus, where the equine influenza genome segment confers on the reassortant virus at least one identifying phenotype of the cold-adapted equine influenza virus.
A cold-adapted equine influenza virus is one that replicates in embryonated chicken eggs at a temperature ranging from about 26xc2x0 C. to about 30xc2x0 C. Preferably, a cold-adapted equine influenza virus, reassortant influenza A virus, or genetically-engineered equine influenza virus of the present invention is attenuated, such that it will not cause disease in a healthy animal.
In one embodiment, a cold-adapted equine influenza virus, reassortant influenza A virus, or genetically-engineered equine influenza virus of the present invention is also temperature sensitive, such that the virus replicates in embryonated chicken eggs at a temperature ranging from about 26xc2x0 C. to about 30xc2x0 C., forms plaques in tissue culture cells at a permissive temperature of about 34xc2x0 C., but does not form plaques in tissue culture cells at a non-permissive temperature of about 39xc2x0 C.
In one embodiment, such a temperature sensitive virus comprises two mutations: a first mutation that inhibits plaque formation at a temperature of about 39xc2x0 C., that mutation co-segregating with the genome segment that encodes the viral nucleoprotein gene; and a second mutation that inhibits all viral protein synthesis at a temperature of about 39xc2x0 C.
In another embodiment, a cold-adapted, temperature sensitive equine influenza virus of the present invention replicates in embryonated chicken eggs at a temperature ranging from about 26xc2x0 C. to about 30xc2x0 C., forms plaques in tissue culture cells at a permissive temperature of about 34xc2x0 C., but does not form plaques in tissue culture cells or express late viral proteins at a non-permissive temperature of about 37xc2x0 C.
Typically, a cold-adapted equine influenza virus of the present invention is produced by passaging a wild-type equine influenza virus one or more times, and then selecting viruses that stably grow and replicate at a reduced temperature. A cold-adapted equine influenza virus produced thereby includes, in certain embodiments, a dominant interference phenotype, that is, the virus, when co-infected with a parental equine influenza virus or heterologous wild-type influenza A virus, will inhibit the growth of that virus.
Examples of cold-adapted equine influenza viruses of the present invention include EIV-P821, identified by accession No. ATCC VR-2625; EIV-P824, identified by accession No. ATCC VR-2624; EIV-MSV+5, identified by accession No. ATCC VR-627; and progeny of such viruses.
Therapeutic compositions of the present invention include from about 105 TCID50 units to about 108 TCID50 units, and preferably about 2xc3x97106 TCID50 units, of a cold-adapted equine influenza virus, reassortant influenza A virus, or genetically-engineered equine influenza virus of the present invention.
The present invention also includes a method to protect an animal from disease caused by an influenza A virus, which includes the step of administering to the animal a therapeutic composition including a cold-adapted equine influenza virus, a reassortant influenza A virus, or a genetically-engineered equine influenza virus of the present invention. Preferred animals to protect include equids, with horses and ponies being particularly preferred.
Yet another embodiment of the present invention is a method to generate a cold-adapted equine influenza virus. The method includes the steps of passaging a wild-type equine influenza virus; and selecting viruses that grow at a reduced temperature. In one embodiment, the method includes repeating the passaging and selection steps one or more times, while progressively reducing the temperature. Passaging of equine influenza virus preferably takes place in embryonated chicken eggs.
Another embodiment is an method to produce a reassortant influenza A virus through genetic reassortment of the genome segments of a donor cold-adapted equine influenza virus of the present invention with the genome segments of a recipient influenza A virus. Reassortant influenza A viruses of the present invention are produced by a method that includes the steps of: (a) mixing the genome segments of a donor cold-adapted equine influenza virus with the genome segments of a recipient influenza A virus, and (b) selecting viruses which include at least one identifying phenotype of the donor equine influenza virus. Identifying phenotypes include cold-adaptation, temperature sensitivity, dominant interference, and attenuation. Preferably, such reassortant viruses at least include the attenuation phenotype of the donor virus. A typical reassortant virus will have the antigenicity of the recipient virus, that is, it will retain the hemagglutinin (HA) and neuraminidase (NA) phenotypes of the recipient virus.
The present invention further provides methods to propagate cold-adapted equine influenza viruses or reassortant influenza A viruses of the present invention. These methods include propagation in embryonated chicken eggs or in tissue culture cells.
The present invention also describes nucleic acid molecules encoding wild-type and cold-adapted equine influenza proteins M, HA, NS, PB2, PB2-N, PB2-C, PB1, PB1-N, PB1-C, and PA-C. One embodiment of the present invention is an isolated equine nucleic acid molecule having a nucleic acid sequence selected from a group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25 SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:106 and SEQ ID NO:108 and a nucleic acid molecule comprising a nucleic acid sequence which is fully complementary to any of such nucleic acid sequences. Another embodiment of the present invention is an isolated equine nucleic acid molecule that encodes a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:81, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:104 and SEQ ID NO:107. Another embodiment is an isolated equine influenza protein that comprises an amino acid sequence selected from a group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:81, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:104 and SEQ ID NO:107. Also included in the present invention is a virus that include any of these nucleic acid molecules or proteins. In one embodiment, such a virus is equine influenza virus or a reassortant virus.
The present invention provides experimentally-generated cold-adapted equine influenza viruses comprising certain defined phenotypes, which are disclosed herein. It is to be noted that the term xe2x80x9caxe2x80x9d or xe2x80x9canxe2x80x9d entity, refers to one or more of that entity; for example, xe2x80x9ca cold-adapted equine influenza virusxe2x80x9d can include one or more cold-adapted equine influenza viruses. As such, the terms xe2x80x9caxe2x80x9d (or xe2x80x9canxe2x80x9d), xe2x80x9cone or more,xe2x80x9d and xe2x80x9cat least onexe2x80x9d can be used interchangeably herein. It is also to be noted that the terms xe2x80x9ccomprising,xe2x80x9d xe2x80x9cincluding,xe2x80x9d and xe2x80x9chavingxe2x80x9d can be used interchangeably. Furthermore, an item xe2x80x9cselected from the group consisting ofxe2x80x9d refers to one or more of the items in that group, including combinations thereof.
A cold-adapted equine influenza virus of the present invention is a virus that has been generated in the laboratory, and as such, is not a virus as occurs in nature. Since the present invention also includes those viruses having the identifying phenotypes of such a cold-adapted equine influenza virus, an equine influenza virus isolated from a mixture of naturally-occurring viruses, i.e., removed from its natural milieu, but having the claimed phenotypes, is included in the present invention. A cold-adapted equine influenza virus of the present invention does not require any specific level of purity. For example, a cold-adapted equine influenza virus grown in embryonated chicken eggs may be in a mixture with the allantoic fluid (AF), and a cold-adapted equine influenza virus grown in tissue culture cells may be in a mixture with disrupted cells and tissue culture medium.
As used herein, an xe2x80x9cequine influenza virusxe2x80x9d is an influenza virus that infects and grows in equids, e.g., horses or ponies. As used herein, xe2x80x9cgrowthxe2x80x9d of a virus denotes the ability of the virus to reproduce or xe2x80x9creplicatexe2x80x9d itself in a permissive host cell. As such, the terms, xe2x80x9cgrowth of a virusxe2x80x9d and xe2x80x9creplication of a virusxe2x80x9d are used interchangeably herein. Growth or replication of a virus in a particular host cell can be demonstrated and measured by standard methods well-known to those skilled in the art of virology. For example, samples containing infectious virus, e.g., as contained in nasopharyngeal secretions from an infected horse, are tested for their ability to cause cytopathic effect (CPE), e.g., virus plaques, in tissue culture cells. Infectious virus may also be detected by inoculation of a sample into the allantoic cavity of embryonated chicken eggs, and then testing the AF of eggs thus inoculated for its ability to agglutinate red blood cells, i.e., cause hemagglutination, due to the presence of the influenza virus hemagglutinin (HA) protein in the AF.
Naturally-occurring, i.e., wild-type, equine influenza viruses replicate well at a temperature from about 34xc2x0 C. to about 39xc2x0 C. For example, wild-type equine influenza virus replicates in embryonated chicken eggs at a temperature of about 34xc2x0 C., and replicates in tissue culture cells at a temperature from about 34xc2x0 C. to about 39xc2x0 C. As used herein, a xe2x80x9ccold-adaptedxe2x80x9d equine influenza virus is an equine influenza virus that has been adapted to grow at a temperature lower than the optimal growth temperature for equine influenza virus. One example of a cold-adapted equine influenza virus of the present invention is a virus that replicates in embryonated chicken eggs at a temperature of about 30xc2x0 C. A preferred cold-adapted equine influenza virus of the present invention replicates in embryonated chicken eggs at a temperature of about 28xc2x0 C. Another preferred cold-adapted equine influenza virus of the present invention replicates in embryonated chicken eggs at a temperature of about 26xc2x0 C. In general, preferred cold-adapted equine influenza viruses of the present invention replicate in embryonated chicken eggs at a temperature ranging from about 26xc2x0 C. to about 30xc2x0 C., i.e., at a range of temperatures at which a wild-type virus will grow poorly or not at all. It should be noted that the ability of such viruses to replicate within that temperature range does not preclude their ability to also replicate at higher or lower temperatures. For example, one embodiment is a cold-adapted equine influenza virus that replicates in embryonated chicken eggs at a temperature of about 26xc2x0 C., but also replicates in tissue culture cells at a temperature of about 34xc2x0 C. As with wild-type equine influenza viruses, cold-adapted equine influenza viruses of the present invention generally form plaques in tissue culture cells, for example Madin Darby Canine Kidney Cells (MDCK) at a temperature of about 34xc2x0 C. Examples of suitable and preferred cold-adapted equine influenza viruses of the present invention are disclosed herein.
One embodiment of the present invention is a cold-adapted equine influenza virus that is produced by a method which includes passaging a wild-type equine influenza virus, and then selecting viruses that grow at a reduced temperature. Cold-adapted equine influenza viruses of the present invention can be produced, for example, by sequentially passaging a wild-type equine influenza virus in embryonated chicken eggs at progressively lower temperatures, thereby selecting for certain members of the virus mixture which stably replicate at the reduced temperature. An example of a passaging procedure is disclosed in detail in the Examples section. During the passaging procedure, one or more mutations appear in certain of the single-stranded RNA segments comprising the influenza virus genome, which alter the genotype, i.e., the primary nucleotide sequence of those RNA segments. As used herein, a xe2x80x9cmutationxe2x80x9d is an alteration of the primary nucleotide sequence of any given RNA segment making up an influenza virus genome. Examples of mutations include substitution of one or more nucleotides, deletion of one or more nucleotides, insertion of one or more nucleotides, or inversion of a stretch of two or more nucleotides. By selecting for those members of the virus mixture that stably replicate at a reduced temperature, a virus with a cold-adaptation phenotype is selected. As used herein, a xe2x80x9cphenotypexe2x80x9d is an observable or measurable characteristic of a biological entity such as a cell or a virus, where the observed characteristic is attributable to a specific genetic configuration of that biological entity, i.e., a certain genotype. As such, a cold-adaptation phenotype is the result of one or more mutations in the virus genome. As used herein, the terms xe2x80x9ca mutation,xe2x80x9d xe2x80x9ca genome,xe2x80x9d xe2x80x9ca genotype,xe2x80x9d or xe2x80x9ca phenotypexe2x80x9d refer to one or more, or at least one mutation, genome, genotype, or phenotype, respectively.
Additional, observable phenotypes in a cold-adapted equine influenza virus may occur, and will generally be the result of one or more additional mutations in the genome of such a virus. For example, a cold-adapted equine influenza virus of the present invention may, in addition, be attenuated, exhibit dominant interference, and/or be temperature sensitive.
In one embodiment, a cold-adapted equine influenza virus of the present invention has a phenotype characterized by attenuation. A cold-adapted equine influenza virus is xe2x80x9cattenuated,xe2x80x9d when administration of the virus to an equine influenza virus-susceptible animal results in reduced or absent clinical signs in that animal, compared to clinical signs observed in animals that are infected with wild-type equine influenza virus. For example, an animal infected with wild-type equine influenza virus will display fever, sneezing, coughing, depression, and nasal discharges. In contrast, an animal administered an attenuated, cold-adapted equine influenza virus of the present invention will display minimal or no, i.e., undetectable, clinical disease signs.
In another embodiment, a cold-adapted equine influenza virus of the present invention comprises a temperature sensitive phenotype. As used herein, a temperature sensitive cold-adapted equine influenza virus replicates at reduced temperatures, but no longer replicates or forms plaques in tissue culture cells at certain higher growth temperatures at which the wild-type virus will replicate and form plaques. While not being bound by theory, it is believed that replication of equine influenza viruses with a temperature sensitive phenotype is largely restricted to the cool passages of the upper respiratory tract, and does not replicate efficiently in the lower respiratory tract, where the virus is more prone to cause disease symptoms. A temperature at which a temperature sensitive virus will grow is referred to herein as a xe2x80x9cpermissivexe2x80x9d temperature for that temperature sensitive virus, and a higher temperature at which the temperature sensitive virus will not grow, but at which a corresponding wild-type virus will grow, is referred to herein as a xe2x80x9cnon-permissivexe2x80x9d temperature for that temperature sensitive virus. For example, certain temperature sensitive cold-adapted equine influenza viruses of the present invention replicate in embryonated chicken eggs at a temperature at or below about 30xc2x0 C., preferably at about 28xc2x0 C. or about 26xc2x0 C., and will form plaques in tissue culture cells at a permissive temperature of about 34xc2x0 C., but will not form plaques in tissue culture cells at a non-permissive temperature of about 39xc2x0 C. Other temperature sensitive cold-adapted equine influenza viruses of the present invention replicate in embryonated chicken eggs at a temperature at or below about 30xc2x0 C., preferably at about 28xc2x0 C. or about 26xc2x0 C., and will form plaques in tissue culture cells at a permissive temperature of about 34xc2x0 C., but will not form plaques in tissue culture cells at a non-permissive temperature of about 37xc2x0 C.
Certain cold-adapted equine influenza viruses of the present invention have a dominant interference phenotype; that is, they dominate an infection when co-infected into cells with another influenza A virus, thereby impairing the growth of that other virus. For example, when a cold-adapted equine influenza virus of the present invention, having a dominant interference phenotype, is co-infected into MDCK cells with the wild-type parental equine influenza virus, A/equine/Kentucky/1/91 (H3N8), growth of the parental virus is impaired. Thus, in an animal that has recently been exposed to, or may be soon exposed to, a virulent influenza virus, i.e., an influenza virus that causes disease symptoms, administration of a therapeutic composition comprising a cold-adapted equine influenza virus having a dominant interference phenotype into the upper respiratory tract of that animal will impair the growth of the virulent virus, thereby ameliorating or reducing disease in that animal, even in the absence of an immune response to the virulent virus.
Dominant interference of a cold-adapted equine influenza virus having a temperature sensitive phenotype can be measured by standard virological methods. For example, separate monolayers of MDCK cells can be infected with (a) a virulent wild-type influenza A virus, (b) a temperature sensitive, cold-adapted equine influenza virus, and (c) both viruses in a co-infection, with all infections done at multiplicities of infection (MOI) of about 2 plaque forming units (pfu) per cell. After infection, the virus yields from the various infected cells are measured by duplicate plaque assays performed at the permissive temperature for the cold-adapted equine influenza virus and at the non-permissive temperature of that virus. A cold adapted equine influenza virus having a temperature sensitive phenotype is unable to form plaques at its non-permissive temperature, while the wild-type virus is able to form plaques at both the permissive and non-permissive temperatures. Thus it is possible to measure the growth of the wild-type virus in the presence of the cold adapted virus by comparing the virus yield at the non-permissive temperature of the cells singly infected with wild-type virus to the yield at the non-permissive temperature of the wild-type virus in doubly infected cells.
Cold-adapted equine influenza viruses of the present invention are characterized primarily by one or more of the following identifying phenotypes: cold-adaptation, temperature sensitivity, dominant interference, and/or attenuation. As used herein, the phrase xe2x80x9can equine influenza virus comprises the identifying phenotype(s) of cold-adaptation, temperature sensitivity, dominant interference, and/or attenuationxe2x80x9d refers to a virus having such a phenotype(s). Examples of such viruses include, but are not limited to, EIV-P821, identified by accession No. ATCC VR-2625, EIV-P824, identified by accession No. ATCC VR-2624, and EIV-MSV+5, identified by accession No. ATCC VR-2627, as well as EIV-MSV0, EIV, MSV+1, EIV-MSV+2, EIV-MSV+3, and EIV-MSV+4. Production of such viruses is described in the examples. For example, cold-adapted equine influenza virus EIV-P821 is characterized by, i.e., has the identifying phenotypes of, (a) cold-adaptation, e.g., its ability to replicate in embryonated chicken eggs at a temperature of about 26xc2x0 C.; (b) temperature sensitivity, e.g., its inability to form plaques in tissue culture cells and to express late gene products at a non-permissive temperature of about 37xc2x0 C., and its inability to form plaques in tissue culture cells and to synthesize any viral proteins at a non-permissive temperature of about 39xc2x0 C.; (c) its attenuation upon administration to an equine influenza virus-susceptible animal; and (d) dominant interference, e.g., its ability, when co-infected into a cell with a wild-type influenza A virus, to interfere with the growth of that wild-type virus. Similarly, cold-adapted equine influenza virus EIV-P824 is characterized by (a) cold adaptation, e.g., its ability to replicate in embryonated chicken eggs at a temperature of about 28xc2x0 C.; (b) temperature sensitivity, e.g., its inability to form plaques in tissue culture cells at a non-permissive temperature of about 39xc2x0 C.; and (c) dominant interference, e.g., its ability, when co-infected into a cell with a wild-type influenza A virus, to interfere with the growth of that wild-type virus. In another example, cold-adapted equine influenza virus EIV-MSV+5 is characterized by (a) cold-adaptation, e.g., its ability to replicate in embryonated chicken eggs at a temperature of about 26xc2x0 C.; (b) temperature sensitivity, e.g., its inability to form plaques in tissue culture cells at a non-permissive temperature of about 39xc2x0 C.; and (c) its attenuation upon administration to an equine influenza virus-susceptible animal.
In certain cases, the RNA segment upon which one or more mutations associated with a certain phenotype occur may be determined through reassortment analysis by standard methods, as disclosed herein. In one embodiment, a cold-adapted equine influenza virus of the present invention comprises a temperature sensitive phenotype that correlates with at least two mutations in the genome of that virus. In this embodiment, one of the two mutations, localized by reassortment analysis as disclosed herein, inhibits, i.e., blocks or prevents, the ability of the virus to form plaques in tissue culture cells at a non-permissive temperature of about 39xc2x0 C. This mutation co-segregates with the segment of the equine influenza virus genome that encodes the nucleoprotein (NP) gene of the virus, i.e., the mutation is located on the same RNA segment as the NP gene. In this embodiment, the second mutation inhibits all protein synthesis at a non-permissive temperature of about 39xc2x0 C. As such, at the non-permissive temperature, the virus genome is incapable of expressing any viral proteins. Examples of cold-adapted equine influenza viruses possessing these characteristics are EIV-P821 and EIV MSV+5. EIV-P821 was generated by serial passaging of a wild-type equine influenza virus in embryonated chicken eggs by methods described in Example 1A. EIV-MSV+5 was derived by further serial passaging of EIV-P821, as described in Example 1E.
Furthermore, a cold-adapted, temperature sensitive equine influenza virus comprising the two mutations which inhibit plaque formation and viral protein synthesis at a non-permissive temperature of about 39xc2x0 C. can comprise one or more additional mutations, which inhibit the virus"" ability to synthesize late gene products and to form plaques in tissue culture cells at a non-permissive temperature of about 37xc2x0 C. An example of a cold-adapted equine influenza virus possessing these characteristics is EIV-P821. This virus isolate replicates in embryonated chicken eggs at a temperature of about 26xc2x0 C., and does not form plaques or express any viral proteins at a temperature of about 39xc2x0 C. Furthermore, EIV-P821 does not form plaques on MDCK cells at a non-permissive temperature of about 37xc2x0 C., and at this temperature, late gene expression is inhibited in such a way that late proteins are not produced, i.e., normal levels of NP protein are synthesized, reduced or undetectable levels of M1 or HA proteins are synthesized, and enhanced levels of the polymerase proteins are synthesized. Since this phenotype is typified by differential viral protein synthesis, it is distinct from the protein synthesis phenotype seen at a non-permissive temperature of about 39xc2x0 C., which is typified by the inhibition of synthesis of all viral proteins.
Pursuant to 37 CFR xc2xa71.802 (a-c), cold-adapted equine influenza viruses, designated herein as EIV-P821, an EIV-P824 were deposited with the American Type Culture Collection (ATCC, 10801 University Boulevard, Manassas, Va. 20110-2209) under the Budapest Treaty as ATCC Accession Nos. ATCC VR-2625, and ATCC VR-2624, respectively, on Jul. 11, 1998. Cold-adapted equine influenza virus EIV-MSV+5 was deposited with the ATCC as ATCC Accession No. ATCC VR-2627 on Aug. 3, 1998. Pursuant to 37 CFR xc2xa71.806, the deposits are made for a term of at least thirty (30) years and at least five (5) years after the most recent request for the furnishing of a sample of the deposit was received by the depository. Pursuant to 37 CFR xc2xa71.808 (a)(2), all restrictions imposed by the depositor on the availability to the public will be irrevocably removed upon the granting of the patent.
Preferred cold-adapted equine influenza viruses of the present invention have the identifying phenotypes of EIV-P821, EIV-P824, and EIV-MSV+5. Particularly preferred cold-adapted equine influenza viruses include EIV-P821, EIV-P824, EIV-MSV+5, and progeny of these viruses. As used herein, xe2x80x9cprogenyxe2x80x9d are xe2x80x9coffspring,xe2x80x9d and as such can slightly altered phenotypes compared to the parent virus, but retain identifying phenotypes of the parent virus, for example, cold-adaptation, temperature sensitivity, dominant interference, or attenuation. For example, cold-adapted equine influenza virus EIV-MSV+5 is a xe2x80x9cprogenyxe2x80x9d of cold-adapted equine influenza virus EIV-P821. xe2x80x9cProgenyxe2x80x9d also include reassortant influenza A viruses that comprise one or more identifying phenotypes of the donor parent virus.
Reassortant influenza A viruses of the present invention are produced by genetic reassortment of the genome segments of a donor cold-adapted equine influenza virus of the present invention with the genome segments of a recipient influenza A virus, and then selecting a reassortant virus that derives at least one of its eight RNA genome segments from the donor virus, such that the reassortant virus acquires at least one identifying phenotype of the donor cold-adapted equine influenza virus. Identifying phenotypes include cold-adaptation, temperature sensitivity, attenuation, and dominant interference. Preferably, reassortant influenza A viruses of the present invention derive at least the attenuation phenotype of the donor virus. Methods to isolate reassortant influenza viruses are well known to those skilled in the art of virology and are disclosed, for example, in Fields, et al., 1996, Fields Virology, 3d ed., Lippincott-Raven; and Palese, et al., 1976, J. Virol., 17, 876-884. Fields, et al., ibid. and Palese, et al., ibid.
A suitable donor equine influenza virus is a cold-adapted equine influenza virus of the present invention, for example, EIV-P821, identified by accession No. ATCC VR-2625, EIV-P824, identified by accession No. ATCC VR-2624, or EIV-MSV+5, identified by accession No. ATCC VR-2627. A suitable recipient influenza A virus can be another equine influenza virus, for example a Eurasian subtype 2 equine influenza virus such as A/equine/Suffolk/89 (H3N8) or a subtype 1 equine influenza virus such as A/Prague/1/56 (H7N7). A recipient influenza A virus can also be any influenza A virus capable of forming a reassortant virus with a donor cold-adapted equine influenza virus. Examples of such influenza A viruses include, but are not limited to, human influenza viruses such as A/Puerto Rico/8/34 (H1N1), A/Hong Kong/156/97 (H5N1), A/Singapore/1/57 (H2N2), and A/Hong Kong/1/68 (H3N2); swine viruses such as A/Swine/Iowa/15/30 (H1N1); and avian viruses such as A/mallard/New York/6750/78 (H2N2) and A/chicken/Hong Kong/258/97 (H5N 1). A reassortant virus of the present invention can include any combination of donor and recipient gene segments, as long as the resulting reassortant virus possesses at least one identifying phenotype of the donor virus.
One example of a reassortant virus of the present invention is a xe2x80x9c6+2xe2x80x9d reassortant virus, in which the six xe2x80x9cinternal gene segments,xe2x80x9d i.e., those comprising the NP, PB2, PB1, PA, M, and NS genes, are derived from the donor cold-adapted equine influenza virus genome, and the two xe2x80x9cexternal gene segments,xe2x80x9d i.e., those comprising the HA and NA genes, are derived from the recipient influenza A virus. A resultant virus thus produced has the attenuated, cold-adapted, temperature sensitive, and/or dominant interference phenotypes of the donor cold-adapted equine influenza virus, but the antigenicity of the recipient strain.
In yet another embodiment, a cold-adapted equine influenza virus of the present invention can be produced through recombinant means. In this approach, one or more specific mutations, associated with identified cold-adaptation, attenuation, temperature sensitivity, or dominant interference phenotypes, are identified and are introduced back into a wild-type equine influenza virus strain using a reverse genetics approach. Reverse genetics entails using RNA polymerase complexes isolated from influenza virus-infected cells to transcribe artificial influenza virus genome segments containing the mutation(s), incorporating the synthesized RNA segment(s) into virus particles using a helper virus, and then selecting for viruses containing the desired changes. Reverse genetics methods for influenza viruses are described, for example, in Enami, et al., 1990, Proc. Natl. Acad. Sci. 87, 3802-3805; and in U.S. Pat. No. 5,578,473, by Palese, et al., issued Nov. 26, 1996. This approach allows one skilled in the art to produce additional cold-adapted equine influenza viruses of the present invention without the need to go through the lengthy cold-adaptation process, and the process of selecting mutants both in vitro and in vivo with the desired virus phenotype.
A cold-adapted equine influenza virus of the present invention may be propagated by standard virological methods well-known to those skilled in the art, examples of which are disclosed herein. For example, a cold-adapted equine influenza virus can be grown in embryonated chicken eggs or in eukaryotic tissue culture cells. Suitable continuous eukaryotic cell lines upon which to grow a cold-adapted equine influenza virus of the present invention include those that support growth of influenza viruses, for example, MDCK cells. Other suitable cells upon which to grow a cold-adapted equine influenza virus of the present invention include, but are not limited to, primary kidney cell cultures of monkey, calf, hamster or chicken.
In one embodiment, the present invention provides a therapeutic composition to protect an animal against disease caused by an influenza A virus, where the therapeutic composition includes either a cold-adapted equine influenza virus or a reassortant influenza A virus comprising at least one genome segment of an equine influenza virus generated by cold-adaptation, wherein the equine influenza virus genome segment confers at least one identifying phenotype of the cold-adapted equine influenza virus. In addition, a therapeutic composition of the present invention can include an equine influenza virus that has been genetically engineered to comprise one or more mutations, where those mutations have been identified to confer a certain identifying phenotype on a cold-adapted equine influenza virus of the present invention. As used herein, the phrase xe2x80x9cdisease caused by an influenza A virusxe2x80x9d refers to the clinical manifestations observed in an animal which has been infected with a virulent influenza A virus. Examples of such clinical manifestations include, but are not limited to, fever, sneezing, coughing, nasal discharge, rates, anorexia and depression. In addition, the phrase xe2x80x9cdisease caused by an influenza A virusxe2x80x9d is defined herein to include shedding of virulent virus by the infected animal. Verification that clinical manifestations observed in an animal correlate with infection by virulent equine influenza virus may be made by several methods, including the detection of a specific antibody and/or T-cell responses to equine influenza virus in the animal. Preferably, verification that clinical manifestations observed in an animal correlate with infection by a virulent influenza A virus is made by the isolation of the virus from the afflicted animal, for example, by swabbing the nasopharyngeal cavity of that animal for virus-containing secretions. Verification of virus isolation may be made by the detection of CPE in tissue culture cells inoculated with the isolated secretions, by inoculation of the isolated secretions into embryonated chicken eggs, where virus replication is detected by the ability of AF from the inoculated eggs to agglutinate erythrocytes, suggesting the presence of the influenza virus hemagglutinin protein, or by use of a commercially available diagnostic test, for example, the Directigen(copyright) FLU A test.
As used herein, the term xe2x80x9cto protectxe2x80x9d includes, for example, to prevent or to treat influenza A virus infection in the subject animal. As such, a therapeutic composition of the present invention can be used, for example, as a prophylactic vaccine to protect a subject animal from influenza disease by administering the therapeutic composition to that animal at some time prior to that animal""s exposure to the virulent virus.
A therapeutic composition of the present invention, comprising a cold-adapted equine influenza virus having a dominant interference phenotype, can also be used to treat an animal that has been recently infected with virulent influenza A virus or is likely to be subsequently exposed in a few days, such that the therapeutic composition immediately interferes with the growth of the virulent virus, prior to the animal""s production of antibodies to the virulent virus. A therapeutic composition comprising a cold-adapted equine influenza virus having a dominant interference phenotype may be effectively administered prior to subsequent exposure for a length of time corresponding to the approximate length of time that a cold-adapted equine influenza virus of the present invention will repliate in the upper respiratory tract of a treated animal, for example, up to about seven days. A therapeutic composition comprising a cold-adapted equine influenza virus having a dominant interference phenotype may be effectively administered following exposure to virulent equine influenza virus for a length of time corresponding to the time required for an infected animal to show disease symptoms, for example, up to about two days.
Therapeutic compositions of the present invention can be administered to any animal susceptible to influenza virus disease, for example, humans, swine, horses and other equids, aquatic birds, domestic and game fowl, seals, mink, and whales. Preferably, a therapeutic composition of the present invention is administered equids. Even more preferably, a therapeutic composition of the present invention is administered to a horse, to protect against equine influenza disease.
Current vaccines available to protect horses against equine influenza virus disease are not effective in protecting young foals, most likely because they cannot overcome the maternal antibody present in these young animals, and often, vaccination at an early age, for example 3 months of age, can lead to tolerance rather than immunity. In one embodiment, and in contrast to existing equine influenza virus vaccines, a therapeutic composition comprising a cold-adapted equine influenza virus of the present invention apparently can produce immunity in young animals. As such, a therapeutic composition of the present invention can be safely and effectively administered to young foals, as young as about 3 months of age, to protect against equine influenza disease without the induction of tolerance.
In one embodiment, a therapeutic composition of the present invention can be multivalent. For example, it can protect an animal from more than one strain of influenza A virus by providing a combination of one or more cold-adapted equine influenza viruses of the present invention, one or more reassortant influenza A viruses, and/or one or more genetically-engineered equine influenza viruses of the present invention. Multivalent therapeutic compositions can include at least two cold-adapted equine influenza viruses, e.g., against North American subtype-2 virus isolates such as A/equine/Kentucky/1/91 (H1N8), and Eurasian subtype-2 virus isolates such as A/equine/Suffolk/89 (H3N8); or one or more subtype-2 virus isolates and a subtype-1 virus isolate such as A/equine/Prague/1/56 (H7N7). Similarly, a multivalent therapeutic composition of the present invention can include a cold-adapted equine influenza virus and a reassortant influenza A virus of the present invention, or two reassortant influenza A viruses of the present invention. A multivalent therapeutic composition of the present invention can also contain one or more formulations to protect against one or more other infectious agents in addition to influenza A virus. Such other infectious agents include, but not limited to: viruses; bacteria; fungi and fungal-related microorganisms; and parasites. Preferable multivalent therapeutic compositions include, but are not limited to, a cold-adapted equine influenza virus, reassortant influenza A virus, or genetically-engineered equine influenza virus of the present invention plus one or more compositions protective against one or more other infectious agents that afflict horses. Suitable infectious agents to protect against include, but are not limited to, equine infectious anemia virus, equine herpes virus, eastern, western, or Venezuelan equine encephalitis virus, tetanus, Streptococcus equi, and Ehrlichia resticii. 
A therapeutic composition of the present invention can be formulated in an excipient that the animal to be treated can tolerate. Examples of such excipients include water, saline, Ringer""s solution, dextrose solution, Hank""s solution, and other aqueous physiologically balanced salt solutions. Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical or biological stability. Examples of buffers include phosphate buffer, bicarbonate buffer, and Tris buffer, while examples of stabilizers include A1/A2 stabilizer, available from Diamond Animal Health, Des Moines, Iowa. Standard formulations can either be liquids or solids which can be taken up in a suitable liquid as a suspension or solution for administration to an animal. In one embodiment, a non-liquid formulation may comprise the excipient salts, buffers, stabilizers, etc., to which sterile water or saline can be added prior to administration.
A therapeutic composition of the present invention may also include one or more adjuvants or carriers. Adjuvants are typically substances that enhance the immune response of an animal to a specific antigen, and carriers include those compounds that increase the half-life of a therapeutic composition in the treated animal. One advantage of a therapeutic composition comprising a cold-adapted equine influenza virus or a reassortant influenza A virus of the present invention is that adjuvants and carriers are not required to produce an efficacious vaccine. Furthermore, in many cases known to those skilled in the art, the advantages of a therapeutic composition of the present invention would be hindered by the use of some adjuvants or carriers. However, it should be noted that use of adjuvants or carriers is not precluded by the present invention.
Therapeutic compositions of the present invention include an amount of a cold-adapted equine influenza virus that is sufficient to protect an animal from challenge with virulent equine influenza virus. In one embodiment, a therapeutic composition of the present invention can include an amount of a cold-adapted equine influenza virus ranging from about 105 tissue culture infectious dose-50 (TCID50) units of virus to about 108 TCID50 units of virus. As used herein, a xe2x80x9cTCID50 unitxe2x80x9d is amount of a virus which results in cytopathic effect in 50% of those cell cultures infected. Methods to measure and calculate TCID50 are known to those skilled in the art and are available, for example, in Reed and Muench, 1938, Am. J. of Hyg. 27, 493-497. A preferred therapeutic composition of the present invention comprises from about 106 TCID50 units to about 107 TCID50 units of a cold-adapted equine influenza virus or reassortant influenza A virus of the present invention. Even more preferred is a therapeutic composition comprising about 2xc3x97106 TCID50 units of a cold-adapted equine influenza virus or reassortant influenza A virus of the present invention.
The present invention also includes methods to protect an animal against disease caused by an influenza A virus comprising administering to the animal a therapeutic composition of the present invention. Preferred are those methods which protect an equid against disease caused by equine influenza virus, where those methods comprise administering to the equid a cold-adapted equine influenza virus. Acceptable protocols to administer therapeutic compositions in an effective manner include individual dose size, number of doses, frequency of dose administration, and mode of administration. Determination of such protocols can be accomplished by those skilled in the art, and examples are disclosed herein.
A preferable method to protect an animal against disease caused by an influenza A virus includes administering to that animal a single dose of a therapeutic composition comprising a cold-adapted equine influenza virus, a reassortant influenza A virus, or genetically-engineered equine influenza virus of the present invention. A suitable single dose is a dose that is capable of protecting an animal from disease when administered one or more times over a suitable time period. The method of the present invention may also include administering subsequent, or booster doses of a therapeutic composition. Booster administrations can be given from about 2 weeks to several years after the original administration. Booster administrations preferably are administered when the immune response of the animal becomes insufficient to protect the animal from disease. Examples of suitable and preferred dosage schedules are disclosed in the Examples section.
A therapeutic composition of the present invention can be administered to an animal by a variety of means, such that the virus will enter and replicate in the mucosal cells in the upper respiratory tract of the treated animal. Such means include, but are not limited to, intranasal administration, oral administration, and intraocular administration. Since influenza viruses naturally infect the mucosa of the upper respiratory tract, a preferred method to administer a therapeutic composition of the present invention is by intranasal administration. Such administration may be accomplished by use of a syringe fitted with cannula, or by use of a nebulizer fitted over the nose and mouth of the animal to be vaccinated.
The efficacy of a therapeutic composition of the present invention to protect an animal against disease caused by influenza A virus can be tested in a variety of ways including, but not limited to, detection of antibodies by, for example, hemagglutination inhibition (HAI) tests, detection of cellular immunity within the treated animal, or challenge of the treated animal with virulent equine influenza virus to determine whether the treated animal is resistant to the development of disease. In addition, efficacy of a therapeutic composition of the present invention comprising a cold-adapted equine influenza virus having a dominant interference phenotype to ameliorate or reduce disease symptoms in an animal previously inoculated or susceptible to inoculation with a virulent, wild-type equine influenza virus can be tested by screening for the reduction or absence of disease symptoms in the treated animal.
The present invention also includes methods to produce a therapeutic composition of the present invention. Suitable and preferred methods for making a therapeutic composition of the present invention are disclosed herein. Pertinent steps involved in producing one type of therapeutic composition of the present invention, i.e., a cold-adapted equine influenza virus, include (a) passaging a wild-type equine influenza virus in vitro, for example, in embryonated chicken eggs; (b) selecting viruses that grow at a reduced temperature; (c) repeating the passaging and selection steps one or more times, at progressively lower temperatures, until virus populations are selected which stably grow at the desired lower temperature; and (d) mixing the resulting virus preparation with suitable excipients.
The pertinent steps involved in producing another type of therapeutic composition of the present invention, i.e., a reassortant influenza A virus having at least one genome segment of an equine influenza virus generated by adaptation, includes the steps of (a) mixing the genome segments of a donor cold-adapted equine influenza virus, which preferably also has the phenotypes of attenuation, temperature sensitivity, or dominant interference, with the genome segments of a recipient influenza A virus, and (b) selecting reassortant viruses that have at least one identifying phenotype of the donor equine influenza virus. Identifying phenotypes to select for include attenuation, cold-adaptation, temperature sensitivity, and dominant interference. Methods to screen for these phenotypes are well known to those skilled in the art, and are disclosed herein. It is preferable to screen for viruses that at least have the phenotype of attenuation.
Using this method to generate a reassortant influenza A virus having at least one genome segment of a equine influenza virus generated by cold-adaptation, one type of reassortant virus to select for is a xe2x80x9c6+2xe2x80x9d reassortant, where the six xe2x80x9cinternal gene segments,xe2x80x9d i.e., those coding for the NP, PB2, PB 1, PA, M, and NS genes, are derived from the donor cold-adapted equine influenza virus genome, and the two xe2x80x9cexternal gene segments,xe2x80x9d i.e., those coding for the HA and NA genes, are derived from the recipient influenza A virus. A resultant virus thus produced can have the cold-adapted, attenuated, temperature sensitive, and/or interference phenotypes of the donor cold-adapted equine influenza virus, but the antigenicity of the recipient strain.
The present invention includes nucleic acid molecules isolated from equine influenza virus wild type strain A/equine/Kentucky/1/91 (H3N8), and cold-adapted equine influenza virus EIV-P821.
In accordance with the present invention, an isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural milieu (i.e., that has been subject to human manipulation) and can include DNA, RNA, or derivatives of either DNA or RNA. As such, xe2x80x9cisolatedxe2x80x9d does not reflect the extent to which the nucleic acid molecule has been purified.
The present invention includes nucleic acid molecules encoding wild-type and cold-adapted equine influenza virus proteins. Nucleic acid molecules of the present invention can be prepared by methods known to one skilled in the art. Proteins of the present invention can be prepared by methods known to one skilled in the art, i.e., recombinant DNA technology. Preferred nucleic acid molecules have coding strands comprising nucleic acid sequences SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO: 64, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:82 , SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:106 and SEQ ID NO:108 and/or a complement thereof Complements are defined as two single strands of nucleic acid in which the nucleotide sequence is such that they will hybridize as a result of base pairing throughout their full length. Given a nucleotide sequence, one of ordinary skill in the art can deduce the complement.
Preferred nucleic acid molecules encoding equine influenza M proteins are neiwtM1023, neiwt1M1023, neiwt2M1023, neiwtM756, neiwt1M756, neiwt2M756, neica1M1023, neica2M1023, neica1M756, and/or neica2M756, the coding strands of which are represented by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, and/or SEQ ID NO:6.
Preferred nucleic acid molecules encoding equine influenza HA proteins are neiwtHA1762, neiwtHA1695, neica1HA1762, neica2HA1762, neica1HA1695, and /or neica2HA1695, the coding strands of which are represented by SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, and/or SEQ ID NO:12.
Preferred nucleic acid molecules encoding equine influenza PB2-N proteins are neiwtPB2-N1241, neiwtPB2-N1241, neica1PB2-N1241 neica2PB2-N1241, neica1PB2-N1214 neica2, and/or PB2-N1214, the coding strands of which are represented by SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, and/or SEQ ID NO:18.
Preferred nucleic acid molecules encoding equine influenza PB2-C proteins are neiwt1PB2-C1233, neiwt2PB2-C1232, neiwtPB2-C1194, neica1PB2-C1232, neica2PB2-C1231, and/or neica1PB2-C1194, the coding strands of which are represented by SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:21, SEQ ID NO:23, and/or SEQ ID NO:25.
Preferred nucleic acid molecules encoding equine influenza PB2 proteins are neiwtPB22341, neiwtPB22277, neica1PB22341, and/or neica1PB22277, the coding strands of which are represented by SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, and/or SEQ ID NO:49.
Preferred nucleic acid molecules encoding equine influenza NS proteins are neiwt1NS891, neiwt2NS891, neiwt1NS690, neiwt2NS690, neiwt3NS888, neiwt3NS690, neiwt4NS468, neiwt4NS293, neica1NS888, neica2NS888, neica1NS690, and/or neica2NS690 the coding strands of which are represented by SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57 and/or SEQ ID NO:59.
Preferred nucleic acid molecules encoding equine influenza PB1-N proteins are neiwt1PB1-N1229, neiwt1PB1N1194, neiwt2PB1-N673, neiwt2PB1-N636, neica1PB1-N1225, neica1PB1-N1185, neica2PB1-N1221, and/or neica2PB1-N1185 the coding strands of which are represented by SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:70, and/or SEQ ID NO:71.
Preferred nucleic acid molecules encoding equine influenza PA-C proteins are neiwt1PA-C1228, neiwt1PA-C1164, neiwt2PA-C1223, neiwt2PA-C1164, neica1PA-C1233, neica2PA-C1233, neica1PA-C1170, and/or neica2PA-C1170 the coding strands of which are represented by SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, and/or SEQ ID NO:82.
Preferred nucleic acid molecules encoding equine influenza PB1-C proteins are neiwt1PB1-C1234, neiwt1PB1-C1188, neiwt2PB1-C1240, neiwt2PB1-C1188, neica1PB1-C1241, neica1PB1-C1188, neica2PB1-C1241 and/or neica2PB1-C1188, the coding strands of which are represented by SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:91, SEQ I) NO:93, SEQ ID NO:94and/or SEQ ID NO:96.
Preferred nucleic acid molecules encoding equine influenza PB1 proteins are neiwtPB12341, neiwtPB12271, neica1PB12341, neica1PB12271, the coding strands of which are represented by SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:106, and/or SEQ ID NO:108.
The present invention includes proteins comprising SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ I) NO:20, SEQ ID NO:24, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO: 81, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, SEQ I) NO:104 and SEQ ID NO: 107 as well as nucleic acid molecules encoding such proteins.
Preferred equine influenza M proteins of the present invention include proteins encoded by a nucleic acid molecule comprising neiwtM1023, neiwt1M1023, neiwt2M1023, neiwtM756, neiwt1M756, neiwt2M756, neica1M1023, neica2M1023, neica1M756, and/or neica2M756. Preferred equine influenza M proteins are PeiwtM252, Peica1M252, and/or Peica2M252. In one embodiment, a preferred equine influenza M protein of the present invention is encoded by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, and/or SEQ ID NO:6, and, as such, has an amino acid sequence that includes SEQ ID NO:2 and/or SEQ ID NO:5.
Preferred equine influenza HA proteins of the present invention include proteins encoded by a nucleic acid molecule comprising neiwtHA1762, neiwtHA1695, neica1HA1762, neica2HA1762, neica1HA1695, and/or neica2HA1695. Preferred equine influenza HA proteins are P PeiwtHA565, Peica1HA565, and/or Peica2HA565. In one embodiment, a preferred equine influenza HA protein of the present invention is encoded by SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, and/or SEQ ID NO:12, and, as such, has an amino acid sequence that includes SEQ ID NO:8 and/or SEQ ID NO:11.
Preferred equine influenza PB2-N proteins of the present invention include proteins encoded by a nucleic acid molecule comprising neiwtPB2-N1241, neiwtPB2-N1214, neica1PB2-N1241 neica2PB2-N1241, neica1PB2-N1214 neica2, and/or PB2-N1214. Preferred equine influenza PB2-N proteins are PwtPB2-N404, Pca1PB2-N404, and/or Pca2PB2-N404. In one embodiment, a preferred equine influenza PB2-N protein of the present invention is encoded by SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, and/or SEQ ID NO:18, and, as such, has an amino acid sequence that includes SEQ ID NO:14 and/or SEQ ID NO:17.
Preferred equine influenza PB2-C proteins of the present invention include proteins encoded by a nucleic acid molecule comprising neiwt1PB2-C1233, neiwt2PB2-C1232, neiwtPB2-C1194, neica1PB2-C1232, neica2PB2-C1231, and/or neica1PB2-C1194. Preferred equine influenza PB2-N proteins are PwtPB2-C398, Pca1PB2-C398, and/or Pca2PB2-C398. In one embodiment, a preferred equine influenza PB2-C protein of the present invention is encoded by SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:21, SEQ ID NO:23, and/or SEQ ID NO:25, and, as such, has an amino acid sequence that includes SEQ ID NO:20 and/or SEQ ID NO:24.
Preferred equine influenza PB2 proteins of the present invention include proteins encoded by a nucleic acid molecule comprising neiwtPB22341, neiwtPB22277, neica1PB22341, and or neica1PB22277. Preferred equine influenza PB2 proteins are PeiwtPB2759,and/or Peica1PB2759. In one embodiment, a preferred equine influenza PB2 protein of the present invention is encoded by SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, and/or SEQ ID NO:49, and, as such, has an amino acid sequence that includes SEQ ID NO:45 and/or SEQ ID NO:48.
Preferred equine influenza NS proteins of the present invention include proteins encoded by a nucleic acid molecule comprising neiwt1NS891, neiwt2NS891, neiwt1NS690, neiwt3NS888, neiwt4NS468, neiwt4NS293, neica1NS888, neica2NS888, and/or neica1NS690. Preferred equine influenza NS proteins are PeiwtNS230, Peiwt4NS97, and/or Peica1NS230. In one embodiment, a preferred equine influenza NS protein of the present invention is encoded by SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57 and/or SEQ ID NO:59, and, as such, has an amino acid sequence that includes SEQ ID NO:51, SEQ ID NO:55 and/or SEQ ID NO:58.
Preferred equine influenza PB1-N proteins of the present invention include proteins encoded by a nucleic acid molecule comprising neiwt1PB1-N1229, neiwt1PB1N1194, neiwt2PB1-N673, neiwt2PB1-N636, neica1PB21-N1225, neica1PB1-N1185, and/or neica2PB1-N1221. Preferred equine influenza PB1-N proteins are Peiwt1PB1-N398, Pwt2PB1-N212, and/or Pca1PB1-N395. In one embodiment, a preferred equine influenza PB1-N protein of the present invention is encoded by SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:70, and/or SEQ ID NO:71, and, as such, has an amino acid sequence that includes SEQ ID NO:63, SEQ ID NO:66 and/or SEQ ID NO:69.
Preferred equine influenza PB1-C proteins of the present invention include proteins encoded by a nucleic acid molecule comprising neiwt1PB1-C1234, neiwt1PB1-C1188, neiwt2PB1-C1240, neiwt2PB1-C1188, neica1PB1-C1241, neica1PB1-C1188, neica2PB1-C1241 and/or neica2PB1-C1188. Preferred equine influenza PB1-C proteins are Peiwt1PB1-C396, Peiwt2PB1-C396 Peica1PB1-C396, and/or Peica2PB1-C396. In one embodiment, a preferred equine influenza PB1-C protein of the present invention is encoded by SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94, and/or SEQ ID NO:96, and, as such, has an amino acid sequence that includes SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, and/or SEQ ID NO:95.
Preferred equine influenza PB1 proteins of the present invention include proteins encoded by a nucleic acid molecule comprising neiwtPB12341, neiwtPB12271, neica1PB12341, neica1PB12271. Preferred equine influenza PB1 proteins are PeiwtPB1757, and/or Peica1PB1757. In one embodiment, a preferred equine influenza PB1 protein of the present invention is encoded by SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:106, and/or SEQ ID NO:108, and, as such, has an amino acid sequence that includes SEQ ID NO:104 and/or SEQ ID NO:107.
Preferred equine influenza PA-C proteins of the present invention include proteins encoded by a nucleic acid molecule comprising neiwt1PA-C1228, neiwt1PA-C1164, neiwt2PA-C1223, neica1PA-C1233, neica2PA-C1233, and/or neica1PA-C1170. Preferred equine influenza PA-C proteins are Peiwt1PA-C388, and/or Peica1PA-C390. In one embodiment, a preferred equine influenza PA-C protein of the present invention is encoded by SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, and/or SEQ ID NO:82, and, as such, has an amino acid sequence that includes SEQ ID NO:77 and/or SEQ ID NO:81.
Nucleic acid sequence SEQ ID NO:1 represents the consensus sequence deduced from the coding strand of PCR amplified nucleic acid molecules denoted herein as neiwt1M1023 and neiwt2M1023, the production of which is disclosed in the Examples. Nucleic acid sequence SEQ ID NO:4 represents the deduced sequence of the coding strand of PCR amplified nucleic acid molecules denoted herein as neica1M1023 and neica2M1023, the production of which is disclosed in the Examples. Nucleic acid sequence SEQ ID NO:7 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as neiwtHA1762, the production of which is disclosed in the Examples. Nucleic acid sequence SEQ ID NO:10 represents the deduced sequence of the coding strand of PCR amplified nucleic acid molecules denoted herein as neica1HA1762 and neica2HA1762, the production of which is disclosed in the Examples. Nucleic acid sequence SEQ ID NO:13 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as neiwtPB2-N1241, the production of which is disclosed in the Examples. Nucleic acid sequence SEQ ID NO:16 represents the deduced sequence of the coding strand of PCR amplified nucleic acid molecules denoted herein as neica1PB2-N1241 and neica2PB2-N1241, the production of which is disclosed in the Examples. Nucleic acid sequence SEQ ID NO:19 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as neiwt1PB2-C1233, the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:22 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as neiwt2PB2-C1232, the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:23 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as neica1PB2-C1232, the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:44 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as neiwtPB22341, the production of which is disclosed in the Examples. Nucleic acid sequence SEQ ID NO:47 represents the deduced sequence of the coding strand of PCR amplified nucleic acid molecules denoted herein as neica1PB22341 the production of which is disclosed in the Examples. Nucleic acid sequence SEQ ID NO:50 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as neiwt1NS891 and neiwt2NS891 the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:53 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as neiwt3NS888, the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:54 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as neiwt4NS468, the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:57 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as neica1NS888 and neica1NS887, the production of which is disclosed in the Examples. Nucleic acid sequence SEQ ID NO:62 represents the deduced sequence of the coding strand of PCR amplified nucleic acid molecules denoted herein as neiwt1PB1-N1229, the production of which is disclosed in the Examples. Nucleic acid sequence SEQ ID NO:65 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as neiwt2PB2-N673, the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:68 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as neica1PB1-N1225, the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:71 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as neica2PB1-N1221, the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:76 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as neiwt1PA-C1228, the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:79 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as neiwt2PA-C1223, the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:80 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as neica1PA-C1233 and neica2PA-C1233 the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:85 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as neica1PB1-C1234 the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:88 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as neiwt2PB1-C1240 the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:91 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as neica1PB1-C1241 the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:94 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as neica2PB1-C1241 the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:103 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as neiwtPB12341 the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:105 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as neiwtPB12271 the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:106 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as neicaPB12341 the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:108 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as neicaPB12271 the production of which is disclosed in the examples. Additional nucleic acid molecules, nucleic acid sequences, proteins and amino acid sequences are described in the Examples.
The present invention includes nucleic acid molecule comprising a cold-adapted equine influenza virus encoding an M protein having an amino acid sequence comprising SEQ ID NO:5. Another embodiment of the present invention includes a nucleic acid molecule comprising a cold-adapted equine influenza virus encoding an HA protein having an amino acid sequence comprising SEQ ID NO:11. Another embodiment of the present invention includes a nucleic acid molecule comprising a cold-adapted equine influenza virus encoding a PB2-N protein having an amino acid sequence comprising SEQ ID NO:17. Another embodiment of the present invention includes a nucleic acid molecule comprising a cold-adapted equine influenza virus encoding a PB2-C protein having an amino acid sequence comprising SEQ ID NO:24. Another embodiment of the present invention includes a nucleic acid molecule comprising a cold-adapted equine influenza virus encoding a PB protein having an amino acid sequence comprising SEQ ID NO:48. Another embodiment of the present invention includes a nucleic acid molecule comprising a cold-adapted equine influenza virus encoding a NS protein having an amino acid sequence comprising SEQ ID NO:58. Another embodiment of the present invention includes a nucleic acid molecule comprising a cold-adapted equine influenza virus encoding a PB1-N protein having an amino acid sequence comprising SEQ ID NO:69. Another embodiment of the present invention includes a nucleic acid molecule comprising a cold-adapted equine influenza virus encoding a PA-C protein having an amino acid sequence comprising SEQ ID NO:81. Another embodiment of the present invention includes a nucleic acid molecule comprising a cold-adapted equine influenza virus encoding a PB1-C protein having an amino acid sequence comprising SEQ ID NO:92. Another embodiment of the present invention includes a nucleic acid molecule comprising a cold-adapted equine influenza virus encoding a PB1 protein having an amino acid sequence comprising SEQ ID NO:107.
It should be noted that since nucleic acid sequencing technology is not entirely error-free, the nucleic acid sequences and amino acid sequences presented herein represent, respectively, apparent nucleic acid sequences of nucleic acid molecules of the present invention and apparent amino acid sequences of M, HA, PB2-N, PB2-C, PB2, NS, PB1-N, PA-C, PB1-C and PB1 proteins of the present invention.
Another embodiment of the present invention is an antibody that selectively binds to an wild-type virus M, HA, PB2-N, PB2-C, PB2, NS, PB1-N, PA-C, PB1-C and PB1 protein of the present invention. Another embodiment of the present invention is an antibody that selectively binds to a cold-adapted virus M, HA, PB2-N, PB2-C, PB2, NS, PB1-N, PA-C, PB1-C and PB1 protein of the present invention. Preferred antibodies selectively bind to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:81, SEQ ID NO:86, SEQ ID NO: 89, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:104 and SEQ ID NO:107.
The following examples are provided for the purposes of illustration and are not intended to limit the scope of the present invention.